Effects of serum on cytotoxicity of nano- and micro-sized ZnO particles

Although an increasing number of in vitro studies are being published regarding the cytotoxicity of nanomaterials, the components of the media for toxicity assays have often varied according to the needs of the scientists. Our aim for this study was to evaluate the influence of serum-in this case, fetal bovine serum-in a cell culture medium on the toxicity of nano-sized (50-70 nm) and micro-sized (<1 μm) ZnO on human lung epithelial cells (A549). The nano- and micro-sized ZnO both exhibited their highest toxicity when exposed to serum-free media, in contrast to exposure in media containing 5 or 10 % serum. This mainly comes not only from the fact that ZnO particles in the serum-free media have a higher dosage-per-cell ratio, which results from large aggregates of particles, rapid sedimentation, absence of protein protection, and lower cell growth rate, but also that extracellular Zn²⁺ release contributes to cytotoxicity. Although more extracellular Zn²⁺ release was observed in serum-containing media, it did not contribute to nano-ZnO cytotoxicity. Furthermore, non-dissolved particles underwent size-dependent particle agglomeration, resulting in size-dependent toxicity in both serum-containing and serum-free media. A low correlation between cytotoxicity and inflammation endpoints in the serum-free medium suggested that some signaling pathways were changed or induced. Since cell growth, transcription behavior for protein production, and physicochemical properties of ZnO particles all were altered in serum-free media, we recommend the use of a serum-containing medium when evaluating the cytotoxicity of NPs.